钛表面固定肝素—多聚赖氨酸纳米颗粒以改善血液相容性的研究
发布时间：2018-10-29 20:55
【摘要】：心血管系统疾病是危害人类健康的重要疾病之一。目前,人工血管、人工心脏瓣膜、血管支架等各种人工器官或医用装置已广泛应用于心血管疾病的治疗,但材料的血液相容性依然存在着严重的问题。为了改善生物材料的血液相容性,本论文通过在钛材料表面构建聚多巴胺涂层,并进一步在涂层上固定多聚赖氨酸(PLL)-肝素(Hep)纳米颗粒,从而提高材料表面的血液相容性。 多巴胺(DM)在碱性条件下能发生自聚合,并可在钛表面实现牢固粘接,形成具有多种官能团(酚羟基、醌基、氨基)的DM薄膜。多聚赖氨酸(PLL)可与肝素(Hep)通过自发静电组装作用形成具有三维结构的纳米颗粒。利用DM与伯氨基可发生麦克尔加成和西弗碱反应的特性,可将含有氨基的Hep/PLL纳米颗粒共价固定至样品表面,构建抗凝功能化表面。利用Zeta电位仪对不同工艺制备的纳米颗粒进行尺寸及Zeta电位值的测定,并进行纳米颗粒制备工艺的优化。通过甲苯胺蓝法对不同工艺纳米颗粒中的肝素进行定量分析；傅里叶变换红外光谱(FTIR)和X射线光电子能谱(XPS)显示纳米颗粒成功地固定到样品表面；通过原子力显微镜(AFM)及水接触角测试进行纳米颗粒固定前后样品表面形貌及亲疏水性变化的表征；利用酸性橙定量氨基、甲苯胺蓝法定量肝素及超微量电子天平对纳米颗粒固定后样品表面氨基含量、肝素含量及颗粒固定总量进行定量表征。 血小板粘附与激活实验表明,钛表面固定的纳米颗粒可显著抑制血小板粘附。纤维蛋白原变性结果表明,固定纳米颗粒后样品表面纤维蛋白原变性程度显著降低,从而进一步降低血小板的聚集和激活。凝血时间检测结果表明纳米颗粒固定后,APTT及TT均有显著延长,但PT对肝素不敏感,检测结果无明显变化。肝素动态释放实验及血小板粘附实验表明,肝素初期释放较快,有助于防止急性凝血发生,后期肝素释放稳定,有利于长期稳定的抗凝。 进一步对材料表面修饰与生物相容性结果的关系研究表明,纳米颗粒的形成受到溶液体系的离子浓度、pH环境以及参与纳米颗粒形成的生物分子的分子量等因素的影响,在模拟正常人体血浆环境的弱碱性PBS中,纳米颗粒能够保持良好的稳定性、均匀性,并发挥较强的抗凝作用。
[Abstract]:Cardiovascular disease is one of the most important diseases that harm human health. At present, artificial blood vessels, artificial heart valves, vascular stents and other artificial organs or medical devices have been widely used in the treatment of cardiovascular diseases, but there are still serious problems in the blood compatibility of materials. In order to improve the blood compatibility of biomaterials, a poly (dopamine) coating was constructed on the surface of titanium, and the poly-lysine (PLL) heparin (Hep) nanoparticles were further immobilized on the coating. So as to improve the surface of the material blood compatibility. Dopamine (DM) can be self-polymerized under alkaline conditions, and can be bonded firmly on titanium surface to form DM films with various functional groups (phenolic hydroxyl, quinone, amino). Poly-lysine (PLL) can interact with heparin (Hep) to form three-dimensional nanoparticles by spontaneous electrostatic assembly. Based on the characteristic that DM can react with primary amino group by Michael addition and sieverine, the Hep/PLL nanoparticles containing amino groups can be covalently immobilized on the surface of the sample to construct an anticoagulation-functionalized surface. The size and Zeta potential of the nanoparticles prepared by different processes were measured by Zeta potentiometer, and the preparation process of nanoparticles was optimized. Quantitative analysis of heparin in nanoparticles with different processes was carried out by toluidine blue method. Fourier transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS) showed that the nanoparticles were successfully immobilized on the surface of the samples. The surface morphology and hydrophobicity of the samples were characterized by atomic force microscope (AFM) (AFM) and water contact angle measurements before and after the nanoparticles were immobilized. The surface amino content, heparin content and total amount of particle fixation were quantitatively characterized by acid orange quantitative amino group, toluidine blue quantitative heparin and ultramicro electron balance. Platelet adhesion and activation test showed that titanium nanoparticles could significantly inhibit platelet adhesion. The results of fibrinogen denaturation showed that the degree of fibrinogen denaturation on the surface of the samples decreased significantly after the nanoparticles were fixed, which further reduced the aggregation and activation of platelet. The results of coagulation time test showed that both APTT and TT were significantly prolonged after immobilization of nanoparticles, but PT was not sensitive to heparin, and the results showed no obvious change. The dynamic release of heparin and platelet adhesion test showed that the initial release of heparin was faster, which was helpful to prevent the occurrence of acute coagulation, and to stabilize the release of heparin in the later stage, which was beneficial to long-term stable anticoagulation. Further studies on the relationship between surface modification and biocompatibility showed that the formation of nanoparticles was influenced by ionic concentration of solution system, pH environment and molecular weight of biomolecules involved in the formation of nanoparticles. In the weakly alkaline PBS which simulates the normal human plasma environment, the nanoparticles can maintain good stability, uniformity and play a strong anticoagulant effect.
【学位授予单位】：西南交通大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：TB383.1;TB324
本文编号：2298798
[Abstract]:Cardiovascular disease is one of the most important diseases that harm human health. At present, artificial blood vessels, artificial heart valves, vascular stents and other artificial organs or medical devices have been widely used in the treatment of cardiovascular diseases, but there are still serious problems in the blood compatibility of materials. In order to improve the blood compatibility of biomaterials, a poly (dopamine) coating was constructed on the surface of titanium, and the poly-lysine (PLL) heparin (Hep) nanoparticles were further immobilized on the coating. So as to improve the surface of the material blood compatibility. Dopamine (DM) can be self-polymerized under alkaline conditions, and can be bonded firmly on titanium surface to form DM films with various functional groups (phenolic hydroxyl, quinone, amino). Poly-lysine (PLL) can interact with heparin (Hep) to form three-dimensional nanoparticles by spontaneous electrostatic assembly. Based on the characteristic that DM can react with primary amino group by Michael addition and sieverine, the Hep/PLL nanoparticles containing amino groups can be covalently immobilized on the surface of the sample to construct an anticoagulation-functionalized surface. The size and Zeta potential of the nanoparticles prepared by different processes were measured by Zeta potentiometer, and the preparation process of nanoparticles was optimized. Quantitative analysis of heparin in nanoparticles with different processes was carried out by toluidine blue method. Fourier transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS) showed that the nanoparticles were successfully immobilized on the surface of the samples. The surface morphology and hydrophobicity of the samples were characterized by atomic force microscope (AFM) (AFM) and water contact angle measurements before and after the nanoparticles were immobilized. The surface amino content, heparin content and total amount of particle fixation were quantitatively characterized by acid orange quantitative amino group, toluidine blue quantitative heparin and ultramicro electron balance. Platelet adhesion and activation test showed that titanium nanoparticles could significantly inhibit platelet adhesion. The results of fibrinogen denaturation showed that the degree of fibrinogen denaturation on the surface of the samples decreased significantly after the nanoparticles were fixed, which further reduced the aggregation and activation of platelet. The results of coagulation time test showed that both APTT and TT were significantly prolonged after immobilization of nanoparticles, but PT was not sensitive to heparin, and the results showed no obvious change. The dynamic release of heparin and platelet adhesion test showed that the initial release of heparin was faster, which was helpful to prevent the occurrence of acute coagulation, and to stabilize the release of heparin in the later stage, which was beneficial to long-term stable anticoagulation. Further studies on the relationship between surface modification and biocompatibility showed that the formation of nanoparticles was influenced by ionic concentration of solution system, pH environment and molecular weight of biomolecules involved in the formation of nanoparticles. In the weakly alkaline PBS which simulates the normal human plasma environment, the nanoparticles can maintain good stability, uniformity and play a strong anticoagulant effect.
【学位授予单位】：西南交通大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：TB383.1;TB324
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